Project Summary Many broadly neutralizing antibodies (bNAbs) recognize structurally discrete sites on the envelope glycoprotein (Env) of human immunodeficiency virus type-1 (HIV-1), such as the CD4-binding site (CD4bs), the strand C of variable regions 1 and 2 (V1V2), the N332 supersite at the base of variable region 3 (V3), and the membrane- proximal external region (MPER). Crystallography and electron microscopy (EM) have revealed how these bNAbs interact with individual epitopes, engineered Env domains, and gp140 trimers, providing a rational basis for vaccine design. The scaffolding method has been used to design novel antigens in hope to elicit epitope- specific, bNAb-like B cell responses. In this R01 application, we will combine the latest structural findings with novel technologies in protein design, B cell engineering and knock-in mouse, and next-generation sequencing (NGS) of B cell repertoire to develop and assess epitope-focused vaccine candidates for three bNAb epitopes. The specific aims (SAs) of our R01 proposal are: (1) to design epitope-focused immunogens for three sites of HIV-1 vulnerability. We hypothesize that the N332 supersite of V3 base, the trimeric V1V2 apex, and MPER can be presented by scaffolds and nanoparticles in their bNAb-bound conformations. In a preliminary study, we have designed a panel of antigens based on various principles such as epitope scaffolding, particulate display, and Fc presentation. We have performed structural and antigenic profiling for a subset of antigens with positive results, and will screen the whole panel of antigens to facilitate rational selection; (2) to assess epitope-focused immunogens in bNAb-presenting B cell lines and knock-in mice. We hypothesize that successfully designed epitope-focused immunogens can activate engineered bNAb-expressing B cells and elicit robust responses in bNAb knock-in mice. Previously, we have developed mouse B cell lines expressing CD4bs-, V1V2- and N332- specific bNAbs and b12 knock-in mouse, which were used to assess rationally designed HIV-1 immunogens. We will develop PGT145-, PGT121/128-, and 10E8-expressing B cell lines and knock-in mice and use these tools to assess epitope-focused immunogens selected in Aim 1; (3) to assess the trimer-prime/epitope-boost strategy and B cell responses in NHPs. We hypothesize that a gp140 trimer will elicit NAbs to diverse epitopes and sequential boosts with epitope-focused immunogens will direct B cell responses to the target epitopes. In our preliminary study, we have tested the epitope-focusing effect and neutralization for selected N332 antigens in C57BL/6 mice. Previously, we have conducted a longitudinal study of B cell responses to an HIV-1 gp140- foldon trimer in non-human primates (NHPs). Here, we will first test the trimer-prime/epitope-boost strategy in mice for different epitopes and then assess the immunogenicity and B cell responses in NHPs. In addition to serological assays, we will use antibody NGS to monitor the temporal B cell responses. Our proposed studies thus constitute an innovative and practical research project to develop epitope-focused HIV-1 vaccines.